Genes that encode proteins that catalyze chemical transformations of alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols may aid in the biosynthesis of energy rich molecules, or in the conversion of such compounds to compounds better suited to specific applications. Such molecules include hydrocarbons (alkane, alkene and isoprenoid), fatty acids, fatty alcohols, fatty aldehydes, esters, ethers, lipids, triglycerides, and waxes, and can be produced from plant derived substrates, such as plant cell walls (lignocellulose, cellulose, hemicellulose, and pectin) starch, and sugar. These molecules are of particular interest as potential sources of energy from biological sources, and thus as possible replacements from energy sources derived from crude oil and its distillates. These molecules are also of interest as potential sources of chemical intermediates, and thus as possible replacements for chemicals derived from crude oil and its distillates.
Yeasts from the genus Candida are industrially important, they tolerate high concentrations of fatty acids and hydrocarbons in their growth media and have been used to produce long chain fatty diacids (Picataggio et al. (1992), Biotechnology (NY): 10, 894-8.) However they frequently lack enzymes that would facilitate conversion of plant cell wall material (cellulose, hemicellulose, pectins and lignins) into sugar monomers for use in biofuel production. Methods for addition of genes encoding proteins capable of catalyzing such conversion into the Candida genome are thus of commercial interest. Further, because yeasts do not always contain enzymatic systems for uptake and metabolism of all of the sugar monomers derived from plant cell wall material, genes encoding enzymes that enable Candida to utilize sugars that it does not normally use, and methods for adding these genes to the Candida genome, are thus of commercial interest.
Currently, α,ω-dicarboxylic acids are almost exclusively produced by chemical conversion processes. However, the chemical processes for production of α,ω-dicarboxylic acids from non-renewable petrochemical feedstocks usually produces numerous unwanted byproducts, requires extensive purification and gives low yields (Picataggio et al., 1992, Bio/Technology 10, 894-898). Moreover, α,ω-dicarboxylic acids with carbon chain lengths greater than 13 are not readily available by chemical synthesis. While several chemical routes to synthesize long-chain α,ω-dicarboxylic acids are available, their synthesis is difficult, costly and requires toxic reagents. Furthermore, most methods result in mixtures containing shorter chain lengths. Furthermore, other than four-carbon α,ω-unsaturated diacids (e.g. maleic acid and fumaric acid), longer chain unsaturated α,ω-dicarboxylic acids or those with other functional groups are currently unavailable since chemical oxidation cleaves unsaturated bonds or modifies them resulting in cis-trans isomerization and other by-products.
Many microorganisms have the ability to produce α,ω-dicarboxylic acids when cultured in n-alkanes and fatty acids, including Candida tropicalis, Candida cloacae, Cryptococcus neoforman and Corynebacterium sp. (Shiio et al., 1971, Agr. Biol. Chem. 35, 2033-2042; Hill et al., 1986, Appl. Microbiol. Biotech. 24: 168-174; and Broadway et al., 1993, J. Gen. Microbiol. 139, 1337-1344). Candida tropicalis and similar yeasts are known to produce α,ω-dicarboxylic acids with carbon lengths from C12 to C22 via an ω-oxidation pathway. The terminal methyl group of n-alkanes or fatty acids is first hydroxylated by a membrane-bound enzyme complex consisting of cytochrome P450 monooxygenase and associated NADPH cytochrome reductase that is the rate-limiting step in the ω-oxidation pathway. Two additional enzymes, the fatty alcohol oxidase and fatty aldehyde dehydrogenase, further oxidize the alcohol to create ω-aldehyde acid and then the corresponding α,ω-dicarboxylic acid (Eschenfeldt et al., 2003, Appl. Environ. Microbiol. 69, 5992-5999). However, there is also a β-oxidation pathway for fatty acid oxidation that exists within Candida tropicalis. Both fatty acids and α,ω-dicarboxylic acids in wild type Candida tropicalis are efficiently degraded after activation to the corresponding acyl-CoA ester through the β-oxidation pathway, leading to carbon-chain length shortening, which results in the low yields of α,ω-dicarboxylic acids and numerous by-products.
Mutants of C. tropicalis in which the β-oxidation of fatty acids is impaired may be used to improve the production of α,ω-dicarboxylic acids (Uemura et al., 1988, J. Am. Oil. Chem. Soc. 64, 1254-1257; and Yi et al., 1989, Appl. Microbiol. Biotech. 30, 327-331). Recently, genetically modified strains of the yeast Candida tropicalis have been developed to increase the production of α,ω-dicarboxylic acids. An engineered Candida tropicalis (Strain H5343, ATCC No. 20962) with the POX4 and POX5 genes that code for enzymes in the first step of fatty acid β-oxidation disrupted was generated so that it can prevent the strain from metabolizing fatty acids, which directs the metabolic flux toward ω-oxidation and results in the accumulation of α,ω-dicarboxylic acids (FIG. 3). See U.S. Pat. No. 5,254,466 and Picataggio et al., 1992, Bio/Technology 10: 894-898, each of which is hereby incorporated by reference herein. Furthermore, by introduction of multiple copies of cytochrome P450 and reductase genes into C. tropicalis in which the β-oxidation pathway is blocked, the C. tropicalis strain AR40 was generated with increased ω-hydroxylase activity and higher specific productivity of diacids from long-chain fatty acids. See, Picataggio et al., 1992, Bio/Technology 10: 894-898 (1992); and U.S. Pat. No. 5,620,878, each of which is hereby incorporated by reference herein. Genes encoding proteins that catalyze chemical transformations of alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols may also reduce the usefulness of these compounds as energy sources, for example by oxidizing them or further metabolizing them. Methods for identifying and eliminating from the Candida genome genes encoding enzymes that oxidize or metabolize alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols are thus of commercial interest. For example fatty alcohols cannot be prepared using any described strain of Candida because the hydroxy fatty acid is oxidized to form a dicarboxylic acid, which has reduced energy content relative to the hydroxy fatty acid. Furthermore, neither the general classes nor the specific sequences of the Candida enzymes responsible for the oxidation from hydroxy fatty acids to dicarboxylic acids have been identified. There is therefore a need in the art for methods to prevent the oxidation of hydroxy fatty acids to diacids during fermentative production.